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DNA viruses: cooperativity and regulation through conformational changes as features of phage assembly.

The assembly of bacteriophages provides experimental model systems for the study of regulation at the level of gene products. We discuss the hypothesis of regulation through sequentially induced conformational changes by which precursor-assemblies become ready at a specific stage of maturation to interact with an additional gene product or nucleic acids. Phage mutants provide excellent experimental model systems for studying, for example, the role and fate of the core in the prehead assembly. The conservative maturation of the prehead to the final, stable head consists of several steps whose complexity reflect that of the virus. It includes packaging of DNA. The surface lattice of maturing preheads apparently undergoes several steps characterized by different conformational states as suggested by in vitro studies on a morphological variant of the prehead, the polyhead of phage T4 (Steven, Couture, Aebi & Showe 1976; Laemmli, Amos & Klug 1976). Addition of a partly purified, enriched proteolytic fraction--which is gene 21-dependent--to empty purified polyheads leads to different conformational states. These seem to go in a direction approaching the structure of the surface of finished capsids as studied by Aebi et al. on gene 24 related (Bijlenga, Aebi & Kellenberger 1976) and other genetically defined giant-variants of T4 phage (Doermann, Eiserling & Boehner 1973). We show some experiments which suggest that high cooperativity is responsible for the stabilization of capsids. The activation energy necessary for dissociation of capsids is very high, 247 kJ for T4 capsids, and 10% smaller for T2. Once the energy barrier has been overcome, the capsids are first structurally modified before they undergo partial and finally complete dissociation.

Capsid↗

Phylogenomics of the nucleosome.

Histones are best known as the architectural proteins that package the DNA of eukaryotic organisms, forming octameric nucleosome cores that the double helix wraps tightly around. Although histones have traditionally been viewed as slowly evolving scaffold proteins that lack diversification beyond their abundant tail modifications, recent studies have revealed that variant histones have evolved for diverse functions. H2A and H3 variants have diversified to assume roles in epigenetic silencing, gene expression and centromere function. Such diversification of histone variants and 'deviants' contradicts the perception of histones as monotonous members of multigene families that indiscriminately package and compact the genome. How these diverse functions have evolved from ancestral forms can be addressed by applying phylogenetic tools to increasingly abundant sequence data.

Amino Acid Sequence↗

Composite co-activator ARC mediates chromatin-directed transcriptional activation.

Gene activation in eukaryotes is regulated by complex mechanisms in which the recruitment and assembly of the transcriptional machinery is directed by gene- and cell-type-specific DNA-binding proteins. When DNA is packaged into chromatin, the regulation of gene activation requires new classes of chromatin-targeting activity. In humans, a multisubunit cofactor functions in a chromatin-selective manner to potentiate synergistic gene activation by the transcriptional activators SREBP-1a and Sp1. Here we show that this activator-recruited cofactor (ARC) interacts directly with several different activators, including SREBP-1a, VP16 and the p65 subunit of NF-kappaB, and strongly enhances transcription directed by these activators in vitro with chromatin-assembled DNA templates. The ARC complex consists of 16 or more subunits; some of these are novel gene products, whereas others are present in other multisubunit cofactors, such as CRSP, NAT and mammalian Mediator. Detailed analysis indicates that the ARC complex is probably identical to the nuclear hormone-receptor cofactor DRIP. Thus, ARC/DRIP is a large composite co-activator that belongs to a family of related cofactors and is targeted by different classes of activator to mediate transcriptional stimulation.

Amino Acid Sequence↗

Fine resolution of the poly ADP-ribosylated domains of polynucleosomal chromatin: DNA gene and integrity analysis; mechanism of histone H1 modification.

The focus of our laboratory has been to ascertain how the poly(ADP-ribosyl)ation reaction influences the structure and biological function of nucleosomal chromatin. Antibody to poly(ADP-ribose (ADP-Rib] was coupled to Sepharose to prepare an immunoaffinity column. The following new information concerning poly(ADP-Rib) and chromatin was obtained with this column: 1) those limiting domains of nucleosomal chromatin undergoing the modification (circa 10%) could be isolated in the "bound" fraction, 2) antibody-bound nucleosomes contained all the poly ADP-ribosylated acceptors and polymerase activity of the bulk chromatin, 3) bound nucleosomes contain significant numbers of internal DNA strand breaks, and [3H]-thymidine (TdR) repair incorporation from in vivo "DNA repair label," compared to the unbound, bulk of the chromatin, 4) the presence of actively transcribed genes in "bound" nucleosomes is being investigated, 5) the acetylation modification of histones occurs in the same domains of chromatin as does poly(ADP-ribosyl)ation, and 6) poly ADP-ribosylated histone H1 can be selectively purified by the immunoaffinity method. These same histone H1 molecules appear to be equally accessible to the histone kinase, phosphorylation modification. In addition, new information has been obtained concerning histone H1 cross-linking by poly(ADP-ribosyl)ation, and on polymerase binding sites to chromatin. We have reconstituted histone H1-depleted chromatin with intact H1 and peptide domains of H1, and subsequently studied H1-poly(ADP-Rib)-complex synthesis. The data indicate that elongation of poly(ADP-Rib) proceeds on the amino terminal region of this histone. By utilizing the new techniques of DNA technology, huge advances in our understanding of the programmed structure of the eukaryotic genome have been accomplished in a relatively short time. To complement this explosion of information, it is important to ascertain how these recently appreciated properties of eukaryotic DNA are packaged within extended and condensed domains of chromatin. The research to be discussed is directed, in part, at this latter topic. We have initiated a program aimed at furthering our comprehension of chromatin structure by studying one specific, enzymatically active chromosomal protein.

Chemical Phenomena↗

A three-dimensional cryo-electron microscopy structure of the bacteriophage phiKZ head.

The three-dimensional structure of the Pseudomonas aeruginosa bacteriophage phiKZ head has been determined by cryo-electron microscopy and image reconstruction to 18A resolution. The head has icosahedral symmetry measuring 1455 A in diameter along 5-fold axes and a unique portal vertex to which is attached an approximately 1800 A-long contractile tail. The 65 kDa major capsid protein, gp120, is organized into a surface lattice of hexamers, with T = 27 triangulation. The shape and size of the hexamers is similar to the hexameric building blocks of the bacteriophages T4, phi29, P22, and HK97. Pentameric vertices of the capsid are occupied by complexes composed of several special vertex proteins. The double-stranded genomic DNA is packaged into a highly condensed series of layers, separated by 24 A, that follow the contour of the inner wall of the capsid.

Capsid Proteins↗

Epigenetics and its implications for plant biology. 1. The epigenetic network in plants.

BACKGROUND: Epigenetics has rapidly evolved in the past decade to form an exciting new branch of biology. In modern terms, 'epigenetics' studies molecular pathways regulating how the genes are packaged in the chromosome and expressed, with effects that are heritable between cell divisions and even across generations. CONTEXT: Epigenetic mechanisms often conflict with Mendelian models of genetics, and many components of the epigenetic systems in plants appeared anomalous. However, it is now clear that these systems govern how the entire genome operates and evolves. SCOPE: In the first part of a two-part review, how epigenetic systems in plants were elucidated is addressed. Also there is a discussion on how the different components of the epigenetic system--regulating DNA methylation, histones and their post-translational modification, and pathways recognizing aberrant transcripts--may work together.

Alleles↗

In vitro packaging of a lambda Dam vector containing EcoRI DNA fragments of Escherichia coli and phage P1.

In this report we describe a coliphage lambda vector system for cloning endo R. EcoRI DNA fragments. This system differs significantly from those previously described in two ways. First, restricted and ligated DNA is encapsidated in vitro. Second, with increasing lambda DNA size in the range 78 to 100% that of wild-type, the efficiency of DNA encapsidation into infectious phage particles markedly increases. For lambda wild-type DNA the efficiency of in vitro packaging (10(6) to 10(7) plaques produced per microgram of added DNA) is equal to, or better than, the standard CaCl2 transfection method. The use of a Dam mutation to facilitate recognition of size classes of inserted fragments is described. Using this vector and in vitro packaging, several E. coli and phage P1 and R.EcoRI fragments were cloned.

Coliphages↗

Primary structure and mapping of the hupA gene of Salmonella typhimurium.

In bacteria, the complex nucleoid structure is folded and maintained by negative superhelical tension and a set of type II DNA-binding proteins, also called histonelike proteins. The most abundant type II DNA-binding protein is HU. Southern blot analysis showed that Salmonella typhimurium contained two HU genes that corresponded to Escherichia coli genes hupA (encoding HU-2 protein) and hupB (encoding HU-1). Salmonella hupA was cloned, and the nucleotide sequence of the gene was determined. Comparison of hupA of E. coli and S. typhimurium revealed that the HU-2 proteins were identical and that there was high conservation of nucleotide sequences outside the coding frames of the genes. A 300-member genomic library of S. typhimurium was constructed by using random transposition of MudP, a specialized chimeric P22-Mu phage that packages chromosomal DNA unidirectionally from its insertion point. Oligonucleotide hybridization against the library identified one MudP insertion that lies within 28 kilobases of hupA; the MudP was 12% linked to purH at 90.5 min on the standard map. Plasmids expressing HU-2 had a surprising phenotype; they caused growth arrest when they were introduced into E. coli strains bearing a himA or hip mutation. These results suggest that IHF and HU have interactive roles in bacteria.

Amino Acid Sequence↗

Nuclear sites of herpes simplex virus type 1 DNA replication and transcription colocalize at early times postinfection and are largely distinct from RNA processing factors.

We have visualized the intracellular localization of herpes simplex virus (HSV) type 1 replication and transcription sites in infected HeLa cells by using direct labelling methods. The number of viral transcription foci increases in a limited way; however, the number of replication sites increases in a near-exponential manner throughout infection, and both replication and transcription sites are found buried throughout the nuclear interior. Simultaneous visualization of viral transcription and replication foci shows that the two processes colocalize at early times, but at later times postinfection, there are additional sites committed solely to replication. This contrasts with the situation in adenovirus-infected cells in which, throughout replication, sites of transcription are adjacent to but do not colocalize with sites of viral DNA replication. The data for an increase in HSV transcription sites suggest an initial phase of replication of input genomes which are then transcribed. Sites of HSV replication colocalize with viral DNA replication and packaging proteins but are largely distinct from the punctate distribution of small nuclear ribonucleoprotein particles. Very high multiplicities of infection have shown an upper limit of some 18 viral transcription foci per nucleus, suggesting cellular constraints on transcription site formation. Use of virus replication mutants confirms that the labelled foci are sites of viral RNA and DNA synthesis; in the absence of viral DNA replication functions, no replication foci and only a limited number of transcription foci were present. Absence of a packaging function had no apparent effect on transcription or replication site formation, illustrating that DNA packaging is not a prerequisite for ongoing DNA synthesis. Further, the essential HSV protein IE63 is required for efficient replication site formation at later times postinfection but is not required for transcription foci formation.

Cell Nucleus↗

A transformation-incompetent, nuclear antigen 2-deleted Epstein-Barr virus associated with replicative infection.

Epstein-Barr virus (EBV) obtained directly from the oropharynx was used to detect viral DNA deleted for the EBV nuclear antigen 2 (EBNA2)-encoding gene that is essential for lymphocyte transformation. By polymerase chain reaction analysis, the deletion was found in virus from 5 of 33 healthy adult donors and 11 of 12 patients with concurrent human immunodeficiency virus infection. Lymphoblastoid cell lines that produce standard transforming EBV also harbored EBNA2-deleted virus in cells permissive of EBV replication. In vitro infectivity studies indicated that the DNA is packaged and transmissible, with biologic properties similar to those of a laboratory mutant, P3HR-1, which also lacks the EBNA2 gene. These findings, obtained from productively infected cell systems, provide evidence for the existence in nature of a transformation-incompetent EBV variant that may facilitate EBV persistence and the emergence of reactivation diseases.

Antigens, Viral↗

A DNA polymerase beta mutant from colon cancer cells induces mutations.

Previous investigations have shown that approximately 35% of the 90 tumors analyzed to date contain mutations within the DNA polymerasebeta (pol beta) gene. The existence of pol beta mutations in a substantial fraction of human tumors studied suggests a link between DNA pol beta and cancer. A DNA pol beta variant, in which Lys-289 has been altered to Met, was identified previously in a colorectal carcinoma. The K289M protein was expressed in mouse L cells containing the lambda cII mutational target. The lambda DNA was packaged and used to infect bacterial cells to obtain the spontaneous mutation frequency. We found that expression of K289M in the mouse cells resulted in a 2.5-fold increase in the mutation frequency. What was most interesting was that expression of K289M in these cells resulted in a 16-fold increase in the frequency of C to G or G to C base substitutions at a specific site within the cII target. By using this cII target sequence, kinetic analysis of the purified K289M protein revealed that it was able to misincorporate dCTP opposite template C and dGTP opposite template G with significantly higher efficiency than the wild-type pol beta protein. We provide evidence that misincorporation of nucleotides by K289M results from altered positioning of the DNA within the active site of the enzyme. Our data are consistent with the interpretation that misincorporation of nucleotides resulting from altered DNA positioning by the K289M protein has the potential to result in tumorigenesis or neoplastic progression.

Base Sequence↗

Encapsidated conformation of bacteriophage T7 DNA.

The structural organization of encapsidated T7 DNA was investigated by cryo-electron microscopy and image processing. A tail-deletion mutant was found to present two preferred views of phage heads: views along the axis through the capsid vertex where the connector protein resides and via which DNA is packaged; and side views perpendicular to this axis. The resulting images reveal striking patterns of concentric rings in axial views, and punctate arrays in side views. As corroborated by computer modeling, these data establish that the T7 chromosome is spooled around this axis in approximately six coaxial shells in a quasi-crystalline packing, possibly guided by the core complex on the inner surface of the connector.

Bacteriophage T7↗

Scaffolding mutants identifying domains required for P22 procapsid assembly and maturation.

Assembly of the icosahedral shells of the dsDNA bacteriophages, herpesviruses, and adenoviruses requires proteins not found in the mature virion, termed scaffolding proteins. The bacteriophage P22 precursor procapsid contains approximately 300 scaffolding molecules within a shell composed of 420 coat protein subunits. Though nonsense mutants are common, few mutants affecting the functions of the scaffolding protein have been recovered. We report here the isolation and characterization of new missense mutants unable to form infectious virions under restrictive conditions. These mutant scaffolding subunits were competent for protein folding and capsid assembly under restrictive conditions. Two mutants were defective in assembly into the procapsid of the portal complex, which serves as the channel through which DNA is packaged. These mutations may identify a region of the scaffolding protein required for interaction with the portal subunits. Two mutants in a different region of the sequence were impaired in scaffolding release from the procapsid both in vivo and in vitro. These mutations may identify a new domain required for scaffolding release. Scaffolding release appeared to be required for capsid expansion; in turn, scaffolding release seemed to depend upon the presence of a portal. This may help to order the pathway of events in phage maturation.

Bacteriophage P22↗

The role of chromatin in transcriptional regulation.

Transcriptional activation is mediated by the facilitated binding of the basal transcription complex to the transcription start site of a promoter. The activation procedure involves protein-protein interactions between specific transcription factors and members of the basal transcription complex. However, since eukaryotic DNA is packaged with histones into nucleosomes the accessibility of the transcription factors is limited. In order to activate transcription, some of the specific transcription factors must have the capacity to bind to their binding sites when organized into nucleosomes. As a next step, the chromatin structure of the promoter needs to be decondensed in order to facilitate the binding of the basal transcription machinery. Recent data have addressed these issues and both binding of transcription factors to their chromatin binding site as well as transcription factor-induced chromatin remodelling have been demonstrated. In addition, factors that are candidates to mediate the chromatin remodelling have recently been identified and characterized. The ability of a transcription factor to recognize its cognate element in a nucleosome is an inheret property that differs among different transcription factors. The implications of the rotational and translational positioning of the DNA within a nucleosome on the accessibility of a transcription factor is described in this review. In addition, nucleosome rearrangement and juxtaposing in the context of transcriptional activation is also discussed.

Animals↗

Comparison of replicative and non-replicative chromatin assembly pathways in HeLa cell extracts.

It has been reported that chromatin assembly in mammalian cell extracts depends exclusively or preferentially on ongoing DNA replication (Stillman, B. (1986) Cell 45, 555-565). More recently, this view has been challenged demonstrating that, in the same extracts, chromatin can also be formed efficiently in the absence of DNA replication (Gruss et al. (1990) EMBO J. 9, 2911-2922). The experiments, described in this communication, were performed to resolve this apparent contradiction. We found that there are at least two distinct in vitro pathways for chromatin assembly in HeLa cell extracts. The replicative pathway requires a nuclear protein, most likely identical with the chromatin assembly factor, described by Stillman (1986, Cell 45, 555-565), and the free soluble histones present in the cytosol of S phase cells. In contrast, a non-replicative pathway was identified that depends on isolated nuclear histones. As one component of the non-replicative assembly pathway we identified a cytosolic factor that was purified to apparent homogeneity and shown to be an acidic 50 kDa polypeptide. The isolated cytosolic 50 kDa protein efficiently promoted nucleosome assembly as demonstrated by one- and two-dimensional gel electrophoresis of in vitro packaged plasmid DNA.

Antigens, Polyomavirus Transforming↗

Structural and functional properties of linker histones and high mobility group proteins in polytene chromosomes.

Variants of histone H1 and high mobility group (HMG) proteins and their genes in Dipteran insects are being studied in our laboratory and have revealed different properties of DNA binding and intrachromosomal distribution. One of the H1 variants of Chironomus is found only in a minority of polytene chromosome bands and differs from the other H1 proteins of the same organism by genomic organization and by an inserted structural motif, the KAPKAP repeat, that is present also in single H1 variants of other, evolutionarily remote organisms. NH2-terminal peptides containing the KAPKAP repeat were found in vitro to interact with DNA, whereas no DNA interaction was observed with the homologous peptide of another H1 variant that does not contain the inserted KAPKAP repeat. We assume that H1 variants containing the KAP motif may interact with a stretch of linker DNA and package chromatin more tightly than other H1 variants. A large series of antibodies directed against different sites in all regions of the H1 molecule is being applied in studying the sites of interaction of the H1 molecule with other molecules in interphase chromatin in terms of antibody epitope accessibility. A search for insect proteins that share properties of the mammalian HMG proteins resulted in isolation and sequencing of two different HMG1 proteins and an HMGI protein. The HMG1 protein of the midge, Chironomus tentans, show a differential distribution in chromosomes. The more abundant cHMG1a protein appears uniformly distributed, whereas the less abundant cHMG1b protein could be localized only in chromosomal puffs. This strongly indicates that these highly similar proteins have different functions in chromatin. The Chironomus HMGI protein and the intron/exon organization of its gene were found to be very similar to human HMGI/Y proteins that are highly abundant in rapidly proliferating cells. Common properties of HMG1 and HMGI proteins include high affinity interaction with AT-rich DNA, irregular DNA structures, and the capacity to bend DNA. These properties suggest that the HMG proteins may have an architectural role in assembling different types of chromatin.

Amino Acid Sequence↗

[Rapid construction of directional cDNA library from human nasopharynx].

OBJECTIVE: To construct a directional cDNA library from human adult nasopharynx by SMART (switching mechanism at 5' end of RNA transcript) technique. METHODS: The total RNA was separated from human adult nasopharynx epithelial tissue and the first-strand cDNA was synthesized through reverse transcription by a modified oligo(dT) primer(contained sfi IB site) while the SMART oligonucleotide(contained sfi IA site) was utilized as a template so that the first-strand cDNA could be extended over the 5' end of mRNA. The double-strand cDNA was amplified by LD-PCR(long-distance PCR) with the above two primers and then digested by sfi I (IA & IB) restriction enzyme. After cDNA size fractionation through CHROMA SPIN column, the double-strand cDNA was ligated into the sfi I-digested lambda TripIEx2 vector and then the recombinant DNA was packaged in vitro. RESULTS: The unamplified human adult nasopharynx cDNA library consists of 1.5 x 10(6) independent clones in which the percentage of recombinant clones is about 100%. The titer of the amplified cDNA library is 3.8 x 10(9) pfu/ml and the average exogenous inserts of the recombinants is 1.5 kb. CONCLUSION: These results shows that the human adult nasopharynx cDNA library has an excellent quality and lays solid foundation for screening and cloning new tumor suppressor genes of nasopharyngeal carcinoma(NPC) and tissue-specific genes of human nasopharynx.

Adult↗

Construction and expression of a recombinant adeno-associated virus that harbors a human beta-globin-encoding cDNA.

Towards a goal of using recombinant adeno-associated viruses (AAV) for the gene therapy of hemoglobinopathies we had previously constructed plasmid pAV h beta G psi 1, which contained a human beta-globin-encoding cDNA (HBB) downstream from the P40 promoter of AAV2 DNA [Ohi et al., Gene 89 (1990) 279-282]. Transfection of the plasmid into human 293 cells (embryonal kidney cell line) resulted in the expression of HBB at the mRNA level as well as rescue and replication of the recombinant AAV genome (Ohi et al., ibid.). The present study demonstrates that the replicated recombinant DNA was packaged into an intact virion by transcomplementation with pAV2 or the defective helpers, pAV delta Bam or pAVXB. The recombinant virus could be isolated by equilibrium CsCl density gradient, the density of which was about 1.4 g/cm3. The defective helpers are used to produce wild-type AAV-free recombinant AAV. The recombinant AAV were infectious and expressed chimeric mRNAs containing the HBB sequence in virus-infected 293, KB (oral epidermoid carcinoma cell line) and K562 (human erythroleukemia cell line) cells. The importance of the infectivity and expression of the recombinant AAV in hematopoietic cells is discussed in the context of gene therapy of hemoglobinopathies.

Blood Cells↗